DESCRIPTION (adapted from the application) Serum transferrin is the protein that transports iron through blood. The protein consists of two similar lobes, with a single iron binding site located at the base of a cleft within each lobe. Under conditions of iron overload, transferrin becomes saturated with iron. However, the rate of iron exchange with low molecular weight ligands tends to be very slow, which severely restricts the ability of therapeutic iron chelating agents to target this readily accessible iron pool. The rate of iron release from transferrin shows a complex dependence on both the ligand concentration and the concentration of inorganic salts in the buffer. For many ligands it appears that the maximum rate of iron release is limited by a slow protein conformational change, but this process is not well understood. Some ligands appear to be able to avoid this limitation either by taking advantage of a separate, first-order pathway that somehow avoids the conformational gating, or by binding at a poorly characterized allosteric anion-binding site that accelerates the rate of the conformational change. The primary objectives of this study are to determine the details of the mechanism of iron exchange between transferrin and low molecular weight ligands and to use this information to design new agents for accelerating iron removal from transferrin under physiological conditions. The proposed work will emphasize studies on recombinant transferrin half-molecules, Tf/2N and Tf/2C, as well as a series of site directed mutants of these molecules. The kinetics of iron release will be followed by visible and fluorescence spectroscopies. Kinetic studies on the native and mutant proteins will characterize the pathways available for iron release. Both computation studies and site directed mutagenesis will be used to locate and characterize the allosteric anion binding site and to determine how anion binding at this site is able to accelerate iron release. New compounds will be synthesized that will be designed to avoid the limitations of the protein conformational change either by accelerate iron release through a separate, first-order pathway or by targeting the allosteric anion binding site to accelerate the rate of the gating conformational change. This is a collaborative project involving faculty with expertise in molecular biology, solution kinetics, organic synthesis, and computational chemistry.